Lecture 04 - Eukaryotic Translation Flashcards
What are proteins
the mini tools/molecular machines that carry out what we term functions in the cell
How much of a mammalians cell energy is used for protein translation
30%
What are the four major stages of protein translation
- load amino acids onto tRNAs
- Initiation
- Elongation
- Termination
Why do tRNAs, elongation factors and termination factors all have similar structures
because they all need to fit in to the A-site
Why is translation initiation more complex in eukaryotes than prokaryotes
it requires more regulation
How many and what initiation factors are required for prokaryotic initiation
three
IF1, IF2, IF3
How many and what initiation factors are required for eukaryotic initiation
nine
eIF1, 2, 3, 4A, 4B, 4C, 4D, 5, 6
What are the four complexes of intitiation
- 43S pre-initiation complex
- eIF4-mRNA complex
- 48S initiation complex
- 80s elongation complex
What occurs during the 43S pre-initiation complex
the complex is put together mRNA
What occurs during the eIF4-mRNA complex
mRNA is prepped so that it is positioned properly when it binds the 43C PIC
What occurs during the 48S initiation complex
the ribosome binds to the mRNA and scans for the start codon
What occurs during the 80S elongation complex
60S is bring in to start elongation
What components make up the 43S pre-initiation complex
40S ribosomal subunit, eIF1 and eIF1A, eIF2-GTP-Met-tRNA, eIF3, eIF5
What is step one of the 43S pre-initiation complex
prepping the ribosome
How is the ribosome prepped
binding of eIF1 and eIF1A causes a conformational change that opens up the channel allowing mRNA access as well as blocking the A and E sites from tRNAs
What site does eIF1A bind to
the A site
What site does eIF1 bind to
the E site
What is the purpose of the eIF2 ternary complex
it delivers the Methionine initiator to the 40S subunit
What is eIF2B
a quanine exchange factor that triggers the reloading of eIF2 with Methionine initiator
What is unique about the methionine initiator
it is structurally different than methionine elongator, they cannot be interchanged, low levels of the initiator reduces translation (regulatory)
What is eIF3
the largest initiation factor (13 subunits), it is used to stabilize the 43S PIC and shields the entry and exit
What is eIF5
a GTPase activating protein that activated eLF2, it is usually blocked to prevent premature hydrolysis of the GTP
What is a UTR
untranslated region
What is the purpose of a 5’UTR
regulation of translation
Where is a UTR
after the 5’ cap and before the Poly-A-tail
What are the three parts of eIF4
eIF4A, eIF4E, eIF4G
What is eIF4A
an RNA helicase that unwinds the mRNA hairpin
What is eIF4E
a cap binding protein
What is eIF4G
the scaffold that binds eIF3 and the polA binding protein
What is eIF4B
a factor that enhances the helicase activity of eIF4A
What is eIF4 used for
the circulation of mRNA
How does eIF4 cause the circulation of mRNA
eIF4G binds both eIF4E and polyA binding protein which leads to the circularization
How is mRNA recruited onto the 43S ribosome subunit
eIF4G interacts with eIF3 (on the ribosome) and eIF4E (the cap binding protein attached to the mRNA)
How does the ribosome find the initiation site
after binding at the 5’ cap it then scans from 5’ to 3’ along the 5’UTR to find the AUG start codon, this must fall into the P-site
What is the Kozak Consensus Sequence
a sequence prior to the start codon that tells the ribosome that the start codon is near
What are the bases in the Kozak Consensus Sequence
GCCRCC(AUG)G
R= either A or G
What does it mean if translation is polycistronic
a weak Kozak Consensus can cause the 48S to scan past the AUG site to the next site causing a different protein to be made from the same mRNA
What is eIF5B
a GTPase that mediate the joining of the 60S and 40S subunits
What change occurs to prep for the addition of the 60S subunit
eIF1 is kicked out during initiation which allows eIF5 to contact eIF2 which hydrolyzes GTP to GDP (it can no longer hold the mRNA in place)
What happens as the 60S subunit binds
the eIFs bound to the 40S subunit dissociate away
What eIF stays bound and why
eIF1A remains bound and acts as a docking site for eIF5B
How does elongation begin
60S subunit stimulates the GTP hydrolysis of eIF5B which dissociates along with eIF1A, the ribosome can now move along the mRNA
How can viruses trick the translation system
fake cap making proteins, cutting caps off host mRNA, hijacking the cap making machinery, structural changes
What is a specific example of a virus tricking the translation system
a type IV virus that uses its RNA to shape like a tRNA, tricks the system into thinking there is something in the P site and triggers translation
How can a virus modify the 40S subunit
it can use its RNA shape to open the mRNA channel without the use of initiation factors
What are the steps of elongation
- eEF1 brings tRNA to the A site, iif the tRNA anticodon matches GTP is hydrolyzed to GDP leaving the tRNA in the A-site
- peptide bond is formed between amino acid and growing chain
- eEF2 fits into the A site and when hydrolyzed slides the ribosome over 3 nucleotides, the now empty tRNA in the E site diffuses away, cycle continues
What are the steps of termination
- eRF1-eRF3 binds to the stop codon
- eRF3 is hydrolyzed and breaks off
- energy is used to kick eRF1 further into the A site which initiates a fake amino transferase
- sometimes the polypeptide just falls off, if not ABCE1 helps kick it off
- ABCE1 pulls apart the subunits by further wedging in eRF1 and acts as a block so the subunits cannot reaggregate
Why do many antibiotics target both prokaryotic and eukaryotic ribosomes
they are similar in structure and function, since the process of translation is so energy draining it is evolutionary conserved if there are big changes made the system will not work
Why do tetracyclines only inhibit prokaryotic translation
it tends to bind to the outer edges of the ribosome which have more ability to change causing more differences between prokaryotic and eukaryotic
How do tetracyclines work
binds to the small subunit and blocks tRNAs from entering the A site
